Methods and vector constructs useful for production of recombinant AAV

ABSTRACT

Methods for efficient production of recombinant AAV employ a host cell containing a first nucleic acid molecule comprising from 5′ to 3′, a parvovirus P5 promoter, a spacer, an AAV rep sequence and an AAV cap gene sequence, wherein said spacer is of sufficient size to reduce expression of the rep78 and rep68 gene products; a second nucleic acid molecule comprising a minigene comprising a transgene flanked by AAV inverse terminal repeats (ITRs) and under the control of regulatory sequences directing expression thereof in a host cell; and helper functions essential to the replication and packaging of rAAV, which functions are not provided by the first or second nucleic acid molecules. Host cells and molecule constructs are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 09/528,470,filed Mar. 17, 2000, now U.S. Pat. No. 6,482,634, which is acontinuation of International Patent Application No. PCT/US98/19479,filed on Sep. 18, 1998, which claims the benefit of the priority of U.S.Patent Application No. 60/059,330, filed on Sep. 19, 1997.

This invention was made with financial assistance from the NationalInstitutes of Health Grant No. NIAMS P01AR/MS43648. The United Statesgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) is a replication-deficient parvovirus, thegenome of which is about 4.6 kb in length, including 145 bp invertedterminal repeats (ITRs). Two open reading frames encode a series of repand cap polypeptides. Rep polypeptides (rep78, rep68, rep62 and rep40)are involved in replication, rescue and integration of the AAV genome.The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking therep and cap open reading frames at the 5′ and 3′ ends are the 145 bpITRs, the first 125 bp of which are capable of forming Y- or T-shapedduplex structures. Of importance for the development of AAV vectors, theentire rep and cap domains can be excised and replaced with atherapeutic or reporter transgene [B. J. Carter, in “Handbook ofParvoviruses”, ed., P. Tijsser, CRC Press, pp.155-168 (1990)] It hasbeen shown that the ITRs represent the minimal sequence required forreplication, rescue, packaging, and integration of the AAV genome.

When this nonpathogenic human virus infects a human cell, the viralgenome integrates into chromosome 19 resulting in latent infection ofthe cell. Production of infectious virus and replication of the virusdoes not occur unless the cell is coinfected with a lytic helper virus,such as adenovirus or herpesvirus. Upon infection with a helper virus,the AAV provirus is rescued and amplified, and both AAV and helper virusare produced The infecting parental ssDNA is expanded to duplexreplicating form (RF) DNAs in a rep dependent manner. The rescued AAVgenomes are packaged into preformed protein capsids (icosahedralsymmetry approximately 20 nm in diameter) and released as infectiousvirions that have packaged either + or − ss DNA genomes following celllysis.

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA to cells. Various groups have studied thepotential use of AAV in the treatment of disease states; however,progress towards establishing AAV as a transducing vector for genetherapy has been slow for a variety of reasons. One obstacle to the useof AAV for delivery of DNA is lack of highly efficient schemes forencapsidation of recombinant genomes and production of infectiousvirions [See, R. Kotin, Hum. Gene Ther., 5:793-801 (1994)].

One proposed solution involves transfecting the recombinantadeno-associated virus (rAAV) containing the transgene into host cellsfollowed by co-infection with wild-type AAV and adenovirus. However,this method leads to unacceptably high levels of wild-type AAV.Incubation of cells with rAAV in the absence of contaminating wild-typeAAV or helper adenovirus is associated with little recombinant geneexpression. In the absence of rep, integration is inefficient and notdirected to chromosome 19.

A widely recognized means for manufacturing transducing AAV virionsentails co-transfection with two different, yet complementing plasmids.One of these contains the therapeutic or reporter transgene sandwichedbetween the two cis acting AAV ITRs. The AAV components that are neededfor rescue and subsequent packaging of progeny recombinant genomes areprovided in trans by a second plasmid encoding the viral open readingframes for rep and cap proteins. However, both rep and cap are toxic tothe host cells. This toxicity has been the major source of difficulty inproviding these genes in trans for the construction of a useful rAAVgene therapy vector.

Other methods have been proposed to enable high titer production ofrAAV. For example, U.S. Pat. No. 5,658,776 refers to packaging systemsand processes for packaging AAV vectors that replace the AAV P5 promoterwith a heterologous promoter. Alternatively, U.S. Pat. No. 5,622,856refers to constructs and methods for AAV vector production, whichprovide constructs formed by moving the homologous P5 promoter to aposition 3′ to the rep genes, and optionally flanking the rep-cap andrepositioned P5 promoter with FRT sequences.

There remains a need in the art for additional methods permitting theefficient production of AAV and recombinant AAV viruses for use inresearch and therapy.

SUMMARY OF THE INVENTION

The present invention provides novel methods, host cells, and vectorconstructs which permit efficient production of rAAV, by decreasing theexpression of the rep78/rep68 gene products, while leaving theexpression of rep52, rep40 and AAV structural proteins at a normallevel.

In one aspect, the invention provides a host cell containing

-   -   (a) a first nucleic acid molecule comprising from 5′ to 3′, a        parvovirus P5 promoter, a spacer, an AAV rep sequence and an AAV        cap sequence, wherein the spacer is of sufficient size to reduce        expression of the rep78 and rep68 gene products;    -   (b) a second nucleic acid molecule comprising a minigene        comprising a transgene flanked by AAV inverse terminal repeats        (ITRs) and under the control of regulatory sequences directing        expression thereof in a host cell; and    -   (c) helper functions essential to the replication and packaging        of rAAV.

In another aspect, the invention provides a nucleic acid molecule usefulin the production of recombinant AAV comprising from 5′ to 3′, ahomologous P5 promoter, a spacer, an AAV rep sequence and an AAV capsequence, wherein the spacer is of sufficient size to reduce, but noteliminate, expression of the rep78 and rep68 gene products.

In yet a further aspect, the invention provides a method for increasingthe production of recombinant adeno-associated virus (rAAV) by culturinga host cell as described above, by which the rep78/rep68 gene productsare reduced in expression, and isolating from the cell lysate or cellculture, high levels of recombinant AAV capable of expressing saidtransgene.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration of a naturally occurring AAV nucleicacid sequence illustrating the P5 promoter 5′ to the start site (ATG) ofthe rep and cap gene sequences.

FIG. 1B is a schematic illustration of a first nucleic acid sequence ofthe present invention showing spacer ‘X’ inserted between the P5promoter and the start site of rep and cap gene sequences.

FIG. 2A is a schematic of plasmid pFG140, a commercially available(Microbix Biosystems, Inc.) plasmid containing a substantial portion ofthe adenovirus type 5 genome except for the E1a and E1b genes. Thisplasmid may be used to provide helper functions in the method of theinvention.

FIG. 2B is a schematic of a smaller plasmid pFΔ13, obtained by digestingpFG140 with RsrII, removing the smaller RsrII fragment and religatingthe plasmid. This plasmid may also be used to provide helper functionsin the method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions for efficientlyproducing high titers of rAAV. The method of this invention may beemployed to produce rAAV carrying therapeutic transgenes, which areparticularly useful in transferring the transgene to a host cell ortissue. These rAAV are also useful as research reagents, or as tools forthe recombinant production of a transgene product in vitro.

I. Compositions

In one embodiment, the invention provides a host cell which contains thefollowing components:

(a) a first nucleic acid molecule comprising from 5′ to 3′, a parvovirusP5 promoter, a spacer, an AAV rep gene sequence and an AAV cap genesequence, wherein the spacer is of sufficient size to reduce expressionof the rep78 and rep68 gene products relative to other rep geneproducts;

(b) a second nucleic acid molecule comprising a minigene comprising atransgene flanked by AAV inverted terminal repeats (ITRs) and under thecontrol of regulatory sequences directing expression thereof in a hostcell; and

(c) helper functions essential to the replication and packaging of rAAV.

A. The First Nucleic Acid Molecule

The key components of the first molecule are arranged in 5′ to 3′ order:the parvovirus P5 promoter, a spacer interposed between the promoter andthe start site of the rep gene sequence, and the cap gene sequence.

The parvovirus P5 promoter used in the first nucleic acid molecule ispreferably homologous to the AAV serotype which provides the rep genesequences and cap gene sequences. Alternatively, the promoter may be aP5 promoter from another AAV type than that which provides the rep andcap sequences. The AAV P5 promoter sequences, as well as the ITRsequences employed in the second nucleic acid molecule described below,may be obtained from any known AAV, including presently identified humanAAV types. Similarly, AAVs known to infect other animals may alsoprovide the P5 promoter, rep and cap gene sequences, and ITRs employedin the constructs of this invention. The selection of the AAV to provideany of these sequences is not anticipated to limit the followinginvention. For example, the P5 promoter may be provided by AAV type 1,AAV type 2, AAV type 3, AAV type 4, AAV type 5, parvovirus type Hl, MVM,LuIII, or from any other parvovirus or AAV serotype. A variety of AAVstrains are available from the American Type Culture Collection or areavailable by request from a variety of commercial and institutionalsources. In the following exemplary embodiments an AAV-2 is used forconvenience.

The spacer is a DNA sequence interposed between the promoter and the repgene ATG (start) site. The spacer may have any desired design; that is,it may be a random sequence of nucleotides, or alternatively, may encodea gene product, such as a marker gene. The spacer may contain geneswhich typically incorporate start/stop and polyA sites. The spacer maybe a non-coding DNA sequence from a prokaryote or eukaryote, arepetitive non-coding sequence, a coding sequence withouttranscriptional controls or coding sequences with transcriptionalcontrols. As illustrated below, two exemplary sources of spacer sequenceare the λ phage ladder sequences or yeast ladder sequences, which areavailable commercially, e.g., from Gibco or Boehringer Mannheim, amongothers.

The spacer may be of any size sufficient to reduce expression of therep78 and rep68 gene products, leaving the rep52, rep40 and cap geneproducts to be expressed at normal levels. The length of the spacer maytherefore range from about 10 bp to about 10.0 kbp. As illustrated belowspacers of 100 bp to about 8.0 kbp in length were used effectively. Inone experimental design, maximum expression levels of rep78 and rep68were achieved with a spacer of about 500 bp in length. Desirably, toreduce the possibility of recombination, the spacer is preferably lessthan 2 kbp in length. However, the invention is not so limited.

The rep gene sequences and cap gene sequences are obtained from the sameor a different serotype of AAV from that which supplies the P5 promoter.These sequences may be contiguous, or may be non-contiguous sequences,as desired, and may be derived from a single AAV or from different AAVsources. The AAV rep and cap sequences, as well as the P5 promoter maybe obtained by conventional means (see Example 1 below). In all cases,in the first nucleic acid molecule, the P5 promoter and spacer are 5′ tothe rep cap sequences.

The first nucleic acid molecule may be in any form which transfers thesecomponents to the host cell. As one example, the first nucleic acidmolecule is preferably in the form of a plasmid, which may contain othernon-viral or viral sequences. However, this molecule does not containthe AAV ITRs and generally does not contain the AAV packaging sequences.As one example, the first nucleic acid molecule described in Example 1below contains a plasmid sequence from the commercially availableBluescript plasmid. A series of such plasmids are identified by thedesignation pJWX-Y, with Y indicating a different size of spacer. Asanother example, a plasmid may contain the key components describedabove, and further contain adenovirus sequences, such as map units 0-1and 9-16 thereof as well as plasmid sequence. This plasmid is desirablyconstructed so that it may be stably transfected into a cell.

Alternatively, the first nucleic acid molecule may be in the form of arecombinant virus, such as an adenovirus or baculovirus. For example,the key components may be inserted as a “minigene” into the E1 region ofan E1-deleted adenovirus vector. See, e.g., published PCT patentapplication Nos. WO96/13598; WO96/13597 and U.S. Pat. No. 5,652,224,among others.

The first nucleic acid molecule may also exist in the host cell as anepisome. Still alternatively, the molecule, or at least the keycomponents described in detail below, may be integrated into thechromosome of the host cell.

The methods employed for constructing a molecule of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in conventional texts. See, e.g., Sambrook etal, Molecular Cloning. A Laboratory Manual. 2d edition, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989). While Example 1provides a specific illustration of the first nucleic acid molecule ofthis invention, using the information provided herein, one of skill inthe art may select and design other suitable first nucleic acidmolecules, with the choice of spacers, P5 promoters and the like, takinginto consideration such factors as length, the presence of at least oneset of translational start and stop signals, and optionally, thepresence of polyadenylation sites.

B. The Second Nucleic Acid Molecule

The second nucleic acid molecule provides in cis a minigene, which isdefined sequences which comprise a selected desired transgene, apromoter, and other regulatory elements necessary for expression of thetransgene in a host cell, flanked by AAV inverse terminal repeats(ITRs).

The AAV sequences employed are preferably the cis-acting 5′ and 3′inverted terminal repeat (ITR) sequences [See, e.g., B J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp.155-168(1990)]. The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in thevectors, although some degree of minor modification of these sequencesis permissible for this use. The ability to modify these ITR sequencesis within the skill of the art. [See, e.g., texts such as Sambrook etal, “Molecular Cloning. A Laboratory Manual.”, 2d edit., Cold SpringHarbor Laboratory, New York (1989); Carter et al, cited above; and K.Fisher et al., J. Virol., 70:520-532 (1996). As described above, the AAVsource of such sequences is not a limitation upon this invention.

In one embodiment, the 5′ and 3′ AAV ITR sequences flank the selectedtransgene sequence and associated regulatory elements (i.e., the 5′ AAVITR is 5′ of the transgene and the regulatory elements and the 3′ AAVITR is 3′ of the transgene and regulatory elements). The transgenesequence of the second molecule is a nucleic acid sequence, heterologousto the AAV sequence, which encodes a polypeptide or protein of interest.The composition of the transgene sequence depends upon the use to whichthe resulting second molecule is to be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include withoutlimitation an E. coli beta-galactosidase (LacZ) cDNA, an alkalinephosphatase gene and a green fluorescent protein gene. These sequences,when associated with regulatory elements which drive their expression,provide signals detectable by conventional means, e.g., ultravioletwavelength absorbance, visible color change, etc. For example, where thetransgene is the LacZ gene, the presence of rAAV is detected by assaysfor beta-galactosidase activity.

However, desirably, the second molecule carries a non-marker gene whichcan be delivered to an animal via the rAAV produced by this method. Apreferred type of transgene sequence is a therapeutic gene whichexpresses a desired gene product in a host cell. These therapeuticnucleic acid sequences typically encode products which, upon expression,are able to correct or complement an inherited or non-inherited geneticdefect or treat an epigenetic disorder or disease. However, the selectedtransgene may encode any product desirable for study. The selection ofthe transgene sequence is not a limitation of this invention.

In addition to the major elements identified above, the minigene of thesecond molecule also includes conventional regulatory elements necessaryto drive expression of the transgene in a cell transfected with thisvector. Thus, the minigene comprises a selected promoter which isoperatively linked to the transgene and located, with the transgene,between the AAV ITR sequences. Selection of the promoter used to driveexpression of the transgene is a routine matter and is not a limitationof the vector.

In a preferred embodiment, the transgene is under the control of acytomegalovirus (CMV) immediate early promoter/enhancer [see, e.g,Boshart et al, Cell, 41:521-530 (1985)]. However, other suitablepromoters may be readily selected by one of skill in the art. Usefulpromoters may be constitutive promoters or regulated (inducible)promoters, which will enable control of expression of the transgeneproduct. Another suitable promoter is the Rous sarcoma virus LTRpromoter/enhancer. Still other promoter/enhancer sequences may beselected by one of skill in the art.

The minigene also desirably contains heterologous nucleic acid sequencesincluding sequences providing signals required for efficientpolyadenylation of the transcript and introns with functional splicedonor and acceptor sites. A common poly-A sequence which is employed inthe exemplary vectors of this invention is that derived from thepapovavirus SV-40. The poly-A sequence generally is inserted followingthe transgene sequences and before the 3′ AAV ITR sequence. A commonintron sequence is also derived from SV-40, and is referred to as theSV-40 T intron sequence. A minigene of the present invention may alsocontain such an intron, desirably located between the promoter/enhancersequence and the transgene. Selection of these and other common vectorelements are conventional and many such sequences are available [see,e.g., Sambrook et al, and references cited therein].

The second nucleic acid molecule carrying the AAV ITRs flanking theminigene may be in any form which transfers these components to the hostcell. As described above for the first nucleic acid molecule, the secondmolecule may contain a plasmid backbone. For example, the second nucleicacid molecule of Example 2 is in the form of a plasmid containing otherviral or non-viral sequences. The plasmid may further contain adenovirussequences, such as map units 0-1 and 9-16.

Alternatively, the second nucleic acid molecule may be in the form of arecombinant virus which is used to infect the host cell. The secondmolecule may be a recombinant replication-defective adenoviruscontaining the transgene operatively linked to expression controlsequences in the region of an adenovirus E1 deletion. Suitable Ad/AAVrecombinant viruses may be produced in accordance with known techniques.See, e.g., International patent applications WO96/13598, published May9, 1996; WO 95/23867 published Sep. 8, 1995, and WO 95/06743 publishedMar. 9, 1995, which are incorporated by reference herein.

As either a plasmid or a virus, the second nucleic acid molecule mayexist in the host cell as an episome or may be integrated into thechromosome of the host cell.

The methods useful for constructing a second nucleic acid molecule ofthis invention are well-known to those of skill in the art and includegenetic engineering, recombinant engineering, and synthetic techniques.See, e.g., Sambrook et al, cited above; and the international patentpublications cited above.

C. Helper Functions

Helper functions essential to the replication and packaging of rAAV arealso provided by or to the host cell in a variety of ways. For example,essential helper functions may be provided by the molecules (a) and (b)which contain, for example, adenovirus gene sequences, as describedabove. As another example, at least one of the molecules (a) or (b) maybe a recombinant virus, which also supplies some or all helper functionsto the cell.

Alternatively, helper functions may be provided by the host cell byvirtue of sequences integrated into the chromosome of the cell. Forexample, the host cell may be an adenovirus or herpesvirus packagingcell, i.e., it expresses adenovirus or herpesvirus proteins useful forthe production of AAV, such as HEK 293 cells and other packaging cells.In the case where the helper functions are expressed by the selectedhost cell, or by the host cell transfected with (a) or (b), noadditional molecules are required.

However, where a packaging cell line is not used as the host cell, orthe helper functions are not sufficiently present, still another sourceof helper functions is a third nucleic acid molecule. In one embodimentthis third nucleic acid molecule is a plasmid which contains helperfunctions. See, for example, the “helper” plasmids of FIG. 2A and FIG.2B, which contain adenovirus sequences in a plasmid backbone.

In another embodiment, the third molecule is a recombinant or wild-typehelper virus, such as an adenovirus, baculovirus, retrovirus orherpesvirus, which provides the helper functions. Whether the optionalthird molecule is a plasmid or virus, it may exist in the cell as anepisome. Where the helper functions are available on a separatemolecule, the “host cell” may be any mammalian cell and not necessarilya packaging cell, such as HEK 293. Examples of suitable parental hostcell lines include, without limitation, HeLa, A549, KB, Detroit, andWI-38 cells. These cell lines are all available from the American TypeCulture Collection, 10801 University Boulevard, Manassa, Va. 20110-2209.Other suitable parent cell lines may be obtained from other sources

Examples 1-3 below illustrate useful molecules and host cells of thisinvention. Using the information provided herein and known techniques,one of skill in the art could readily construct a different recombinantvirus (i.e., non-adenovirus) or a plasmid molecule which is capable ofdriving expression of the selected component in the host cell. Forexample, although less preferred because of their inability to infectnon-dividing cells, vectors carrying the required elements of the firstor second nucleic acid molecules, e.g., the P5-spacer-rep-cap or theITR-transgene-ITR sequences, may be readily constructed using e.g.,retroviruses or baculoviruses. Therefore, this invention is not limitedby the virus or plasmid selected for purposes of introducing theessential elements of the first nucleic acid sequence or second nucleicacid sequence or the optional third nucleic acid sequence into the hostcell.

II. Methods of the Invention

In another embodiment, the present invention provides a method forincreasing the production of rAAV by decreasing the expression of therep78 and rep68 gene products, keeping the expression of rep52 andrep40, and the cap gene products at normal levels. This method includesthe steps of culturing a host cell described above, which containsnucleic acid molecules (a) and (b), and helper functions (c), asdescribed above; and isolating from the cell lysate or cell culture, arecombinant AAV capable of expressing the transgene of molecule (b).

In one embodiment of the method, a selected host cell is co-transfectedwith the first and second nucleic acid molecules, as described above,and then infected with a wild-type (wt) or replication defective virus,or transfected helper plasmid, to supply the helper functions. Suitablehelper viruses may be readily selected by those of skill in the art andinclude, for example, wt Ad2, wt Ad5, and herpesviruses, as well as thereplication defective adenovirus dl309. Suitable helper plasmids mayalso be readily selected by those of skill in the art and include, forexample, the pFG140, pFΔ13, and pBHG10, which are described herein Inanother embodiment, the host cell is an adenovirus packaging cell, suchas HEK 293, and the first or second nucleic acid molecule is arecombinant virus, which also contains the remaining adenovirus helperfunctions necessary to package AAV in the presence of the essentialelements provided by (b) and (c). Selection of the means by which thehelper functions are provided is not a limitation on the presentinvention.

Suitable techniques for introducing the molecules of this invention intothe host cell are known to those of skill in the art. When all moleculesor vectors are present in a cell and the cell is provided with helperfunctions, the rAAV is efficiently produced.

In another embodiment of the method of this invention, a packaging cellline is constructed which stably expresses the helper functions (c), orwhich expresses the first nucleic molecule (a) According to this aspectof the method, the cell line expressing the (c) or (a) elements can besubstituted for the vector or plasmid (a) or (c) as described above.Thus, only the second molecule (i.e., the cis plasmid) described aboveis subsequently introduced into the cell.

Having obtained such a helper-expressing cell line, this cell line canbe infected (or transfected) with the first vector (a) containing therep and cap genes and the second vector (b) containing the minigenedescribed above.

The methods of this invention demonstrate that the limiting step for thehigh yield of rAAV is not the replication of cis plasmid; but thepackaging process and rep78 and rep68 can interfere with the packagingprocess directly or indirectly.

III. Production of Vectors and rAAV

Assembly of the selected DNA sequences contained within each of themolecules described above may be accomplished utilizing conventionaltechniques. Such techniques include cDNA cloning such as those describedin texts [Sambrook et al, cited above], use of overlappingoligonucleotide sequences of the adenovirus, AAV genome combined withpolymerase chain reaction, synthetic methods, and any other suitablemethods which provide the desired nucleotide sequence.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan. Where appropriate, standard transfection and co-transfectiontechniques may be employed, e.g., CaPO₄ transfection or electoporationusing the human embryonic kidney (HEK) 293 cell line (a human kidneycell line containing a functional adenovirus E1a gene which provides atransacting E1a protein). Other conventional methods employed in thisinvention include homologous recombination of the viral genomes,plaquing of viruses in agar overlay, methods of measuring signalgeneration, and the like.

Following infection/transfection, the host cell is then cultured toenable production of the rAAV [See, e.g., F. L. Graham and L. Prevec,Methods Mol. Biol., 7:109-128 (1991), incorporated by reference herein].Desirably, once the rAAV is identified, it may be recovered and purifiedusing standard techniques.

The following examples illustrate the preferred methods of theinvention. These examples are illustrative only and are not intended tolimit the scope of the invention.

EXAMPLE 1 Construction of First Nucleic Acid Molecules

A. Trans Plasmids

An exemplary first molecule of the present invention is provided as aplasmid containing the P5—spacer—AAV rep and cap genes as follows. SeeFIGS. 1A and 1B.

The AAV P5 promoter was amplified from the 121 bp XbaI-BamHI fragmentfrom plasmid psub201, which contains the entire AAV2 genome [R. J.Samulski et al, J. Virol., 61:3096-3101 (1987)] by PCR using twooligonucleotides:

-   -   oligo 1: TGT AGT TAA TGA TTA ACC CGC CAT GCT ACT TAT C [SEQ ID        NO: 2] and oligo 2: GGC GGC TGC GCG TTC AAA CCT CCC GCT TCA AAA        TG [SEQ ID NO: 3]. This P5 promoter sequence was subsequently        cloned into plasmid pCR2.1 (Invitrogen), resulting in a new        plasmid, pCR-P5. The AAV rep and cap coding region is amplified        from the AAV type 2 virus by primers TATTTAAGCCCGAGTGAGCT [SEQ        ID NO: 4] and TAGCATGGCGGGTTAATCATTAACTACA [SEQ ID NO: 5] and        cloned into the unique SmaI site of pBluescript (Promega). The        resulting plasmid is called pBS-AAV.

The P5 promoter is then excised from pCR-P5 by digestion with BamHI andXhoI, filled in by Klenow and then cloned into the ClaI site of plasmidpBS-AAV. The resulting plasmid, designated as P5-X, contains a uniqueEcoRV site between the P5 promoter and the initiation codon of rep78.

The helper plasmid (i.e., first nucleic acid molecule) is made bycloning the desired spacer, in this case, either the λ phage or yeast100 bp ladder and 500 bp ladder sequences (Gibco; BRL) into the EcoRVsite in P5-X. The resulting series of plasmids are designated as pJWX-Y(FIG. 1A). Reference to Table I codifies these plasmids as pJWX-Y, inwhich Y indicates the size of the plasmid. The spacer sizes present inthese plasmids range from 100 bp to 8 kb. FIG. 1A represents the normalrelationship of P5 to the rep and cap genes. FIG. 1B represents theP5-spacer-rep-cap configuration of the first nucleic molecule.

EXAMPLE 2 Construction of Second Nucleic Acid Molecule

A “cis” plasmid, or second nucleic acid molecule useful in the presentinvention contains a minigene comprising AAV ITRs flanking a promoterand transgene, the minigene inserted into a plasmid backbone. In thepresent example, the exemplary cis plasmid is AV.CMVLacZ [SEQ ID NO: 1;see International Patent Application NO. WO95/13598] was utilized as thecis plasmid (the second nucleic acid molecule) useful in the methods ofthis invention. It is a rAAV cassette in which AAV rep and cap genes arereplaced with a minigene expressing β-galactosidase from a CMV promoter.The linear arrangement of pAV.CMVLacZ includes:

(a) the 5′ AAV ITR (bp 1-173) obtained by PCR using pAV2 [C. A. Laughlinet al, Gene, 23: 65-73 (1983)] as template [nucleotide numbers 365-538of SEQ ID NO:1];

(b) a CMV immediate early enhancer/promoter [Boshart et al, Cell,41:521-530 (1985); nucleotide numbers 563-1157 of SEQ ID NO: 1],

(c) an SV40 intron (nucleotide numbers 1178-1179 of SEQ ID NO:1),

-   -   (d) E. coli beta-galactosidase cDNA (nucleotide numbers        1356-4827 of SEQ ID NO: 1),

(e) an SV40 polyadenylation signal (a 237 BamHI-BclI restrictionfragment containing the cleavage/poly-A signals from both the early andlate transcription units; nucleotide numbers 4839-5037 of SEQ ID NO: 1)and

(f) 3′AAV ITR, obtained from pAV2 as a SnaBI-BglII fragment (nucleotidenumbers 5053-5221 of SEQ ID NO:1). The remainder of the plasmid issimply plasmid backbone from a pBR322-derivative.

EXAMPLE 3 Production of rAAV

According to one embodiment of the present invention, 5×10⁶ HEK 293cells (American Type Culture Collection, Rockville, Md.) weretransfected as follows: 2 μg of the helper plasmid pFΔ13 (FIG. 2B), 1 μgcis plasmid (pAV.CMVLacZ [SEQ ID NO: 1] of Example 2) and 1 μg of atrans plasmid selected from the groups listed in Table I, weretransfected into 293 cells using DOTAP (Boehringer Mannheim Biotech).

Forty-eight hours later, each group of cells were harvested [J. Price etal, Proc. Natl. Acad. Sci., USA, 84:156-160 (1987)]. The cell lysate wasthen subjected to three rounds of freeze-thaw cycles. The amount of rAAVvirus in supernatant was then titer by x-gal assay. To get pure rAAVvirus, the cell lysate can be purified by CsCl gradient.

Table I lists the identity of the first nucleic acid molecule (i.e., thetrans plasmid), the size of the spacer therein, and the total yield ofrAAV from 2×10⁷ cells in two production experiments was reported asLacZ-forming units (LFU). In Table I below 1 unit represent 1×10⁵ LFU.In this case, pAdAAV represents a helper plasmid containing no spacerbetween P5 and repcap (see FIG. 1A).

TABLE I Total Yield pTrans plasmid Spacer 1st 2nd Avg. pAdAAV none 0.62.6 1.6 pJWX-100  100 bp 1 8.7 4.9 pJWX-200  2 × 100 bp repeats 22 33.428 pJWX-300  3 × 100 bp repeats 25 70 48 pJWX-400  4 × 100 bp repeats 2960 45 pJWX-500  5 × 100 bp repeats 24 55 40 pJWX-600  6 × 100 bp repeats31 130 81 pJWX-700  7 × 100 bp repeats 26 31.4 29 pJWX-800  8 × 100 bprepeats 29 28.3 29 pJWX-900  9 × 100 bp repeats N/A 24 24 pJWX-1 k 10 ×100 bp repeats N/A 33 33 pJWX-1.1 k 11 × 100 bp repeats 15 29 22pJWX-0.5 k  500 bp insert 30 72 51 pJWX-1 k   1 kb insert 25 40 33pJWX-1.5 k  1.5 kb insert 16 19 18 pJWX-2 k   2 kb insert 20 21 21pJWX-2.5 k  2.5 kb insert 19 32 26 pJWX-3 k 3.02 kb insert 20 17 19pJWX-3.5 k  3.5 kb insert 21 18.4 20 pJWX-4 k 4.01 kb insert 26 12.7 19pJWX-4.5 k  4.5 kb insert 16 25 21 pJWX-5 k 5.01 kb insert 25 13.2 19pJWX-8 k   8 kb insert 27 17.4 22

Surprisingly the rAAV yield is greatly improved using these helperplasmids.

EXAMPLE 4 Further Characterization of the rAAV Produced by the Method

A. Western Blots

A Western blot was performed on transfections using a variety ofdifferent trans plasmids having different spacer sequences (Example 3).The results showed that the expression of rep78 and rep68 from theserAAV was greatly reduced while the expression of rep52, rep40 and AAVstructural proteins remained unchanged. The results showed thatreplication of the gene containing cis plasmid was not significantlyaffected even though the amount of rep78 and rep68 was decreased.

Therefore, the optimization of rep78 and rep68 expression is criticalfor high titer rAAV production.

B. Southern Blot

A Southern blot was performed on transfections using the method of thisinvention using as “trans” plasmids or first nucleic acid molecules,either mock (no trans plasmid), AdAAV (a plasmid containing no spacerbetween the P5 and rep ATG site), or trans plasmids of the presentinvention containing varying sizes of spacer.

The position of the dimer and monomer bands did not change across alltrans plasmids used. This demonstrates that the spacers between the P5promoter and rep and cap genes of the trans plasmids of the invention donot affect the replication of the cis plasmid in the method. The AAVrep78168 expression is sufficient in the method of this invention tomaintain normal AAV replication.

EXAMPLE 5 Titer Comparisons

The following two tables of data represent studies in which the methodsof the present invention were performed by, (a) transfecting 293 cellsby calcium phosphate precipitation with the trans plasmids identified ineach table, the cis plasmid of Example 2 and the pFΔ13 helper plasmid ofFIG. 2A or (b) transfecting 5×10⁶ HEK 293 cells (American Type CultureCollection, Manassas, Va.) with 1 μg cis plasmid (pAV.CMVLacZ [SEQ IDNO: 1] of Example 2) and 1 μg of a trans plasmid selected from thegroups listed in Table III using Lipofectin (Gibco BRL). These cellswere thereafter infected with wildtype adenovirus type 5 at an MOI of 5to supply the helper functions.

Table II illustrates the virus titers of three trials using protocol(a).

TABLE II Trans Plasmid Trial 1 Trial 2 Trial 3 Mock (no trans) 0 0 0AdAAV (no spacer) 100 100 100 pJWX-23 693 391 838 pJWX-4k 344 330 444pJWX-1k 441 321 475 pJWX-500 344 278 437

Table III illustrates the titers of one trial using protocol (b). Inthis case a titer unit of 1 is equivalent to 3×10⁶ infectious particles.

TABLE III Trans plasmid Titer Mock 0.0 AdAAV 0.9 pJWX-100 3.4 pJWX-2002.4 pJWX-300 5.4 pJWX-400 3.9 pJWX-500 7.2 pJWX-600 3.6 pJWX-700 2.3pJWX-800 3.0 pJWX-900 3.6 pJWX-1k 2.5 pJWX-1.5k 1.0 pJWX-2k 2.0

Publications cited in this specification are incorporated herein byreference. Numerous modifications and variations of the presentinvention are included in the above-identified specification and areexpected to be obvious to one of skill in the art. Such modificationsand alterations to the processes of the present invention are believedto be encompassed in the scope of the claims appended hereto.

1. A recombinant host cell containing (a) a first nucleic acid moleculecomprising a parvovirus P5 promoter in its native configuration withresponse to wild-type AAV, an AAV rep gene sequence, an AAV cap genesequence, and a space of sufficient size to reduce expression of therep78 and rep68 gene products relative to other rep gene products,wherein said spacer is heterologous to said promoter and is interposedbetween said promoter and the start site of the rep gene; (b) a secondnucleic acid molecule comprising a minigene comprising a transgeneflanked by AAV inverted terminal repeats (ITRs) and under the control ofregulatory sequences directing expression thereof in a host cell; and(c) helper functions essential to the replication and packaging of rAAV;wherein the nucleic acid of (a) or (b) is selected from the groupconsisting of a plasmid, a recombinant virus, and an episome.
 2. Thecell according to claim 1 wherein said spacer sequence is between about10 bp to 10 kb in length.
 3. The cell according to claim 1 wherein saidspacer sequence is between 100 bp to 3.8 kb in length.
 4. The cellaccording to claim 1 wherein said spacer sequence is about 500 bp inlength.
 5. The cell according to claim 1 wherein said spacer sequence isa random sequence of nucleotides.
 6. The cell according to claim 1wherein said spacer sequence encodes a gene product.
 7. The cellaccording to claim 1 wherein said first nucleic acid molecule isselected from the group consisting of a plasmid, a recombinant virus,and an episome.
 8. The cell according to claim 1 wherein said firstnucleic acid molecule is integrated into the chromosome of said cell. 9.The cell according to claim 1 wherein said parvovirus P5 promoter isfrom a strain of AAV selected from the group consisting of AAV type 1,AAV type 2, AAV type H1.
 10. The cell according to claim 1 wherein saidrep gene sequences are from AAV type 1 or AAV type
 5. 11. The cellaccording to claim 1 wherein said second nucleic acid molecule isselected from the group consisting of a plasmid, a recombinant virus,and an episome.
 12. The cell according to claim 1 wherein said secondnucleic acid molecule is integrated into the chromosome of said cell.13. The cell according to claim 1 wherein said helper functions areprovided by a third nucleic acid molecule.
 14. A recombinant host cellcontaining (a) a first nucleic acid molecule comprising parvovirus P5promoter in its native configuration with respect to wild-type AAV, anAAV rep gene sequence, an AAV cap gene sequence, and a spacer ofsufficient size to reduce expression of the rep78 and rep68 geneproducts relative to other rep gene products, wherein said spacer isheterologous to said promoter and is interposed between said promoterand the start site of the rep gene; (b) a second nucleic acid moleculecomprising a minigene comprising a transgene flanked by AAV invertedterminal repeats (ITRs) and under the control of regulatory sequencesdirecting expression thereof in a host cell; and (c) helper functionsessential to the replication and packaging of rAAV provided by a thirdnucleic acid molecule selected from the group consisting of a plasma, arecombinant or wild-type virus, and an episome.
 15. The cell accordingto claim 13 wherein said third nucleic acid molecule is integrated intothe chromosome of said cell.
 16. The cell according to claim 1 which isderived from a HEK 293 cell.
 17. A method for producing recombinantadeno-associated virus (AAV), said method comprising the steps of (a)culturing a recombinant host cell according to claim 1; and (b)isolating from said cell or cell culture, a recombinant AAV capable ofexpressing said transgene.